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We used four complementary techniques to investigate the presence of oxytocin peptide in the hypophysis and brain of the snake Bothrops jararaca. A high-pressure liquid chromatographic analysis failed to show oxytocin in extracts of hypophysial and brain tissues but provided estimative values of the amounts of vasotocin (12 ng/mg hypophysis and 0.5 ng/mg brain) and mesotocin (500 pg/mg hypophysis and 8 pg/mg brain). Western blots with a polyclonal anti-oxytocin antibody failed to detect oxytocin in both tissues but detected compounds with higher molecular weight than oxytocin, as well as a relatively weak cross-reactivity with mesotocin. The reverse transcription-polymerase chain reaction analysis failed to detect the expression of oxytocin gene transcript, but detected a transcript related to the mesotocin-neurophysin precursor in both tissues. Immunohistochemistry with the same anti-oxytocin antibody detected strong staining in the neurohypophysis and in few fibers in the inner zone of the median eminence, which was not abolished by pre-adsorption of this antibody with oxytocin, vasopressin, vasotocin or mesotocin and might not be attributed to oxytocin. In conclusion, our data demonstrate the absence of oxytocin in the central nervous system of the snake B. jararaca and underline the pitfalls that can result from the use of a single technique to investigate the presence of peptides in tissues.